High copy number plasmids and their derivatives

ABSTRACT

This invention provides origins of replication capable of amplifying nucleic acid at an increased copy number within a cell. In particular, the invention provides origins of replication that amplify plasmid to an increased copy number within a bacterium.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisionalapplication No. 60/407,053, filed Aug. 29, 2002, which is incorporatedby reference herein.

BACKGROUND OF THE INVENTION

[0002] Plasmids are commonly used as vectors for the cloning andexpression of foreign genes in bacteria. It is particularly desirable,for this purpose, to use plasmids that are present in high copy number,either in order to obtain the foreign DNA in a large quantity, or inorder to increase the amount of expressed product.

[0003] The production of large quantities of proteins for use astherapeutics, additives, and other myriad applications remains achallenge. Large-scale fermentation is a commonly used method, but isexpensive and difficult to maintain the required quality and consistencyof product. When producing proteins in bacteria, vectors that have ahigh copy number are generally sought because the amount of protein isoften directly proportional to gene dosage.

[0004] DNA vaccination, or DNA-mediated immunization, refers to thedirect introduction into a living species of plasmid or non-plasmid DNAor RNA that can cause expression of antigenic protein(s) or peptide(s)in the newly transfected cells. The nucleic acid may be introduced intotissues of the host species by variety of techniques, e.g., needleinjection, particle bombardment or orally using various DNAformulations, which may be either “naked” DNA, coated microparticles, orliposomes or biodegradable microcapsules or microspheres.

[0005] Runaway replication plasmid vectors have been developed forexpression of genes in bacteria. While these runaway-replication plasmidvectors have been used to produce a variety of proteins, including hGCSFand somatotropin, the amount of protein produced has been limited bysuch factors as the copy number, and cell death resulting from runawayreplication, thus preventing the use of continuous fermentationtechniques. Thus, there is a need for expression vector systems withoutthese limitations.

[0006] Plasmids are extrachromosomal circular DNA molecules that aretransferable from one bacterium to another and replicate independentlyof the bacterial chromosome. A given plasmid can be present in a highcopy number inside a bacterial cell. The copy number is a geneticcharacteristic of each plasmid. For example, in the ColE1-type plasmids(such as plasmids of the families pBR, pUC, and the like), the copynumber is under the control of a DNA region within the replicationorigin of the plasmid (ORI) which extends approximately between bases2940 and 3130 (numbering of the bases of pBR322 proposed by Peden (Gene22:277-280, 1983). A portion of this region, situated between bases 2970and 3089, is transcribed into RNAs called RNAI and RNAII. RNAI, inparticular, is thought to play a role in the regulation of the plasmidcopy number.

[0007] The RNAII species provides an RNA primer which forms a complex ator near the origin from which DNA synthesis is initiated; the RNAIspecies interferes with the formation of this initiation complex(Tomizawa, Cell 47:89-97, 1986; and Lin-Chao & Cohen, Cell 65:1233-1242,1991. Transcription of the two RNA species is controlled by separatepromoter sequences associated with the DNA sequences that encode thetranscripts (for reviews, see, e.g., Eguchi et.al. Biochemistry60:631-652, 1991; and Polisky, Cell 55:929-932, 1988). In addition,there is a small polypeptide (the rop protein) that is believed tointeract with the promoter for RNAII. The polypeptide is not essentialfor replication, however.

[0008] The origin of replication and the RNA coding sequences and theirassociated promoters together provide an internally self-regulatedsystem that controls the replication incompatibility (as describedbelow) and the copy number of these plasmids. Certain other plasmids,exemplified by RI and some Staphyloccocal plasmids, also controlreplication initiation at the transcriptional level, but by a messengerRNA species whose product provides an initiation factor, probably apolypeptide, which is involved in DNA replication.

[0009] Plasmids carrying a mutation that influences the copy number havebeen described in the art. For example, Boros et al. (Gene 30:257-260,1984) describe a mutant plasmid derived from pBR322. The copy number ofthis plasmid per cell is increased by about 200-fold relative to thecopy number of pBR322. The increase in the number of copies results froma G to T transversion at position 3075 on the 2846-3363 HinfI fragment,close to the 3′ end of the sequence that encodes RNAI. It had been shownpreviously that the same mutation in the ColE1 plasmid ColE1, which hasa replication origin similar to that of pBR322, also increases the copynumber of the plasmid (up to 300 per cell). See, e.g, Muesing et al.,Cell, 24:235-242, 1981.

[0010] Recent advances have demonstrated the importance of regulation ofthe RNAI and RNAII species in controlling copy number of ColE1-derivedplasmids. Several factors affect the decay of RNAI including RNase E,polynucleotide phosphorylase, poly(A) polymerase (see, e.g., Xu et.al.Proc. Natl. Acad. Sci. 90:6756-6760, 1993) and RNase III (e.g., Binnie,et.al. Microbiology 145:3089-3100, 1999).

[0011] RNase E is a single strand endonuclease that cleaves RNAI nearits 5′ end and converts it to an unstable pRNAI₅, which relievesreplication repression (see, e.g., Lin-Chao & Cohen, Cell 65:1233-1242,1991). Mutations in the pcnB gene, which encodes poly(A) phosphorylase(PAP I), cause prolongation of the half-life of RNAI, and decrease thecopy number of ColE1-type plasmids. PAP I adds adenosine residues to the3′ end of RNAI, which accelerates its degradation (Xu et.al., Proc.Natl. Acad. Sci. 90:6756-6760, 1993). Alteration of the enzymaticactivity of these enzymes can potentially affect copy number.Furthermore, alterations in the RNAI or RNAII species themselves maychange their recognition profile for any or all of these enzymes.Further, it was also noted that the lengths of RNAI or RNAII affecttheir hybridization to one another (Tomizawa, Cell 47:89-97, 1986), solength of these RNAs could also be a determinant of copy number. Thus,mutations within the origin of replication that significantly alter thethree dimensional conformation of RNAI or RNAII may have dramaticaffects on their half-lives, interaction with one another, andultimately plasmid copy number.

[0012] Several cloning vectors are derivatives of the ColE1-relatedplasmid pMB1, including pBR322 (Bolivar, et.al. Gene 2:95-113, 1997),and high-copy versions in the pUC series [e.g., Viera & Messing Gene19:259-268, 1982; Yanisch-Perron, et.al. Gene 33:103-119, 1985) andpBluescript (Stratagene, La Jolla, Calif.). Plasmids that are compatiblewith pMB1 include those that use the p25A-related origins of replication(Bartolome et.al., Gene 102:75-78, 1991). In general, the copy number ofthese plasmids is between 15-20 copies per chromosome. While medium tolow copy number vectors may be suitable for many applications, their usecan be limiting when high levels of expression of a gene, or multiplegenes, is required. Although replication of ColE1-like plasmids isdependent on DNA polymerase I and is regulated by the interaction ofRNAI and RNAII transcripts, distinct incompatibility groups have beenidentified (see, e.g., Selzer, et.al. Cell 32 :119-129, 1983; and Som &Tomizawa, Mol. Gen. Genet. 187:375-383, 1982). In this regard, thesegregation properties of plasmids within a cell are controlled bysequences in the origin of replication for ColE1 (Bedbrook, et.al.Nature 281:447-452, 1979). Regions of the ColE1 origin of replicationcritical for compatibility have been identified (see, e.g.,Hashimoto-Gotoh & Inselburg, J. Bacteriol. 139:608-619, 1979). TheColE1-like plasmid RSF1030, for example, is able to reside with bothpMB1 and p15A-derived plasmids, as well as with non-ColE1vectors such aspSC101. Additionally, a high copy variant of pRSF1030 with a singlenucleotide change has recently been described ([Phillips, et.al.Biotechniques 28:400-408, 2000). Thus, changes within the origin ofreplication may alter the compatiblity phenotype of a given plasmid.However, there is a need for additional high copy number plasmids.

SUMMARY OF THE INVENTION

[0013] The present invention provides origins of replication capable ofamplifying nucleic acid at an increased copy number within a cell. Inparticular, the invention provides origins of replication capable ofamplifying nucleic acid at an increased copy number within a prokaryoticcell, preferably a bacterium. The basis of the invention is thediscovery that an insertion, a deletion, a substitution or a combinationthereof in defined regions of the origin of replication result in a veryhigh copy number and can also regulate compatibility. Preferably, theorigins of replication are present on a circular polynucleotide such asa plasmid vector. Additionally, the invention provides for a cellcontaining one or more of said origins of replication and providesmethods for producing the plasmids, genes, and gene products derivedtherefrom.

[0014] In one embodiment, the invention provides a plasmid that grows toa higher copy number (e.g., at least 2-fold) relative to parentalplasmids. The plasmid comprises at least one mutation, e.g., aninsertion, deletion, or substitution, in the origin of replication of aColE1-type plasmid. Such mutations typically occur within the regiondefined as positions 1 to 210 as determined with reference to SEQ IDNO:1. In some embodiment, the mutation is within the region defined aspositions 1 to 150, as determined with reference to SEQ ID NO:1. Thedeletion, insertion, or substitution may involve one or more positions.The deletions can be of any length, e.g., 1 to 150 base pairs, but aretypically less than 100 base pairs. Similarly, the insertions may be ofany length, but are typically from 1 to 100 base pairs. Substitution canoccur at one ore more positions, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 15, 20, 30, 40, 50, or 100 positions. Additionally, multiplemutations and combinations of substitutions, insertions and/or deletionscan also be present in an origin of replication of the invention.

[0015] Often, mutations, e.g., deletions, occur in the region of theorigin encoding RNAI. Deletion mutants typically comprise deletions ofvarying numbers of nucleotides, e.g., from 1 to 70 nucleotides, and mostoften, deletions of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,25, 30, 35, 40, 45, or 50 nucleotides. Similarly, insertion mutantstypically comprise insertions of varying numbers of nucleotides, e.g.,from 1 to 70 nucleotides, and most often, insertions of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50nucleotides.

[0016] Exemplary substitutions, deletions, or insertions can occur atthe following positions: positions 1 to 68, positions 40 to 50,positions 57 to 60, positions 25 to 27, positions 59-64, positions192-194, positions 128-134, positions 126-128, positions 127-129,positions 61-62, positions 93-103, positions 47-51, positions 59-65, andpositions 58-63. In some embodiments, an origin of the inventioncomprises a sequences set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, or SEQ ID NO:13.

[0017] In some embodiments, e.g., SEQ ID NO:5 or SEQ ID NO:11, a plasmidcomprising an origin of replication with a mutation as described hereinis compatible with other colE1-like origins and therefore can be used ina single cell with a second plasmid that comprises a different colE1origin, e.g., a parent colE1-type origin such as that of pBluescript.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows a schematic of RNA regulation of ColE1-type originsof replication (thick black bar). RNAII is produced from a promotor P,and is transcribed as a sense strand. This RNA species is utilized byDNA polymerase I as a primer for DNA synthesis during initiation ofreplication. Control of replication is mediated by RNAI, which istranscribed in the antisense direction from a promotor P, and whichmediates suppression of replication by binding to RNAII and causing aconformational change in the primer that causes inefficient extension byDNA polymerase.

[0019]FIG. 2 shows the sequence of a ColE1-related origin of replicationfrom the pBluescript plasmid. The residues are from 1158 to 1825 of thefull length plasmid. The residues encoding RNAII are in upper case andthe residues encoding RNAI are underlined.

[0020]FIG. 3 shows the sequence of DNA that encodes an RNAII moleculethat can prime synthesis of DNA from a ColE1-type plasmid.

[0021]FIG. 4 shows an ori5′ mutant multiple sequence alignment. Themutants are indicate by the number designation at the left. Thesemutations confer a high-copy number phenotype. The RNA II region isindicated in capital letters. The RNAI region is blackened. Deletionsare indicated by dashes. Sequence differences in ori mutant 4.1 areindicated by underlined residues.

[0022]FIG. 5 provides exemplary data that show the change in copy numberfrom origin of replication mutant 3.4 (right, labeled “evolved plasmid”)compared to wild-type pBluescript plasmid (left).

[0023]FIG. 6 depicts the structure of the RNAII region of pBluescript.Positions of various ColE1 deletion mutants are indicated by the orireference number and shown as solid lines. Ori2.2 is not included inthis figures, as the mutation occurs outside of the RNAII region.

DETAILED DESCRIPTION

[0024] The present invention provides origins of replication capable ofamplifying nucleic acid to an increased copy number within a cell,typically a prokaryotic cell such as a bacterium. Preferably, theorigins of replication are present on a circular polynucleotide such asa plasmid vector. Additionally, the invention provides a cell containingone or more of the origins of replication of the invention. In thisrespect, the present invention provides origins of replication onplasmids that not only have increased copy number, but also have alteredcompatibilities with other plasmids. The invention also provides methodsfor producing the plasmids, genes, and gene products derived therefrom.

[0025] Definitions

[0026] The terms “origin” or “origin of replication” as used hereinrefer to a sequence of nucleic acid that will allow its replicationwithin a cell, or in a cell free extract containing nucleic acidpolymerase.

[0027] The term “ColE1-type”, “ColE1-related”, or “ColE1-derived” originof replication refers to a member of a family of related origins ofreplication that have control features similar to ColE1. ColE1-relatedorigins as defined herein are at least 70% identical, often 80%identical and typically 90% identical to SEQ ID NO:1. “ColE1-type”plasmids encode an RNAII primer that is used by DNA polymerase toinitiate replication, and an RNAI molecule that regulates initiationthrough antisense interaction on RNAII. Most often ColE1-type plasmidsreplicate with a theta-type mechanism. Examples of ColE1-related originsare well known in the art and include, for example, the origins ofreplication of plasmids pMB 1, pBR322, the pUC series, p15A and RSF1030(see, e.g., Selzer et.al., Cell 32:119-129, 1983). “ColE1-related”origins may be compatible or incompatible with one another.

[0028] A “high copy number plasmid” as used herein refers to a plasmidthat comprises an origin of replication that results in an increase inplasmid copy number of at least 2-fold, often, 5- or 10-fold, incomparison to a control plasmid comprising the origin of replication setforth in SEQ ID NO:1

[0029] The term “compatible” as applied to plasmids refers to two ormore plasmids that can exist stably together in a single cell formultiple generations. “Incompatible” plasmids are unable to bemaintained stably together in a single cell for multiple generations.

[0030] The term “nucleoside” refers to a molecule comprising thecovalent linkage of a pyrimidine or purine to a pentose ring (such asribose or deoxyribose).

[0031] The term “nucleotide” refers to the phosphate ester of anucleoside.

[0032] The term “nucleic acid” is used interchangeably with“polynucleotide” to refer to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs). Unless otherwise indicated, a particularnucleic acid sequence also implicitly encompasses complementarysequences, as well as the sequence explicitly indicated.

[0033] The term “position” as it relates to a nucleic acid sequencerefers to the location of a given residue in the polynucleotide chain,not to the number of residues in a sequence per se. For example,“position” in a polynucleotide sequence is defined as the location of anucleotide in the polynucleotide chain with reference to at least oneother nucleotide. The phrase “determined with reference to” in thecontext of identifying changes in a nucleic acid sequence means that thenucleotide at a particular position of the reference sequence is deletedor inserted. For example, in SEQ ID NO:3, the first ten nucleotides ofthe sequence are GGTTTGTTTG. As determined with reference to SEQ IDNO:1, these nucleotides are at positions 69-78. Thus, the origin ofreplication set forth in SEQ ID NO:3 has a deletion of nucleotides 1-68,relative to SEQ ID NO: 1.

[0034] The term “nucleotide deletion” as applied to a polynucleotidemeans that a polynucleotide has had one or more specific residuesremoved from one or more positions in the polynucleotide chain when theresulting polynucleotide is compared to the parental or other referencesequence.

[0035] The term “nucleotide insertion” or “nucleotide addition” meansthat a polynucleotide has had specific residues added to thepolynucleotide chain, such that at least one of the original residuesnow occupies a new position in the polynucleotide when compared to theparental or other reference sequence.

[0036] The term “nucleotide substitution” as applied to a polynucleotidemeans that a nucleotide at a position of a nucleic acid sequence hasbeen substituted when compared to the parental or other referencesequence.

[0037] A “subsequence” used with respect to a nucleic acid sequencerefers to a segment of the nucleic acid sequence that is less than thefull-length nucleic acid sequence.

[0038] The term “DNA” refers to deoxyribonucleic acid. It will beunderstood by those of skill in the art that where manipulations aredescribed herein that relate to DNA they will also apply to RNA.

[0039] The term “circular DNA” as used herein refers to a nucleic acidin which no double-stranded DNA ends are present. A circular DNA may besingle-stranded or double-stranded and further may, comprisesingle-stranded DNA ends. For example, a circular DNA will be present ifsingle-stranded DNA ends exist but hydrogen bonding keeps the twostrands of the double-stranded molecule hybridized to one another suchthat a double-stranded DNA end is not created by the presence of twosingle-stranded ends in proximity to one another. Such a circulardouble-stranded polynucleotide is often referred to as “nicked”.Examples of circular DNA molecules include plasmids and phagemids.

[0040] The term “random” or “random position” as applied to apolynucleotide refers to a process by which any of the specific residuepositions may be selected. Random as used herein does not mean that allpoints or point of cleavage or nucleotides or positions are selected orchosen with equal frequency. Rather random focuses on the unpredictablenature of the process, i.e. the worker cannot predict a priori where anevent will occur or what position any base will have. Finally, not allpositions need be available for cleavage for the process to be random asto the available positions or bases. For example, a polynucleotide witha length of N may have any or all of its positions (i.e. 1, 2, . . . N)affected by a manipulation. In the addition (insertion) or deletion ofresidues, a polynucleotide necessarily must have covalent bonds (such asphosphodiester bonds) cleaved, thereafter which residues are deleted oradded (i.e. the total number of positions is decreased or increased,respectively). In describing “deletions at random positions” in apolynucleotide of length N, it is meant that any or all of the N (in acircular polynucleotide) or N−1 (in a linear polynucleotide) covalentlinkages between nucleotides (i.e. phosphodiester bonds) are broken, andat least one nucleotide at the end is removed prior to re-ligation.Thus, in a process causing “deletions at random positions” the finallength of the polynucleotide (N, or the number of positions) necessarilydecreases. Similarly, In describing “insertions at random positions” ina polynucleotide of length N, it is meant that any or all of the N (in acircular polynucleotide) or N−1 (in a linear polynucleotide) covalentlinkages between nucleotides (i.e. phosphodiester bonds) are broken, andat least one new nucleotide (i.e. a new position) is added at the endprior to re-ligation. Thus, in a process causing “insertions at randompositions” the final length of the polynucleotide (N, or the number ofpositions) necessarily increases. It is recognized that a combination ofprocesses involving “deletions at random positions” and “insertions atrandom positions” may allow the final length of the polynucleotide toremain unchanged (i.e. the additions cancel out the deletions and thefinal number of positions remains the same, however the nucleotidesoccupying the positions may be different). In describing “randomcleavage” or a “single random break” in a polynucleotide of length N, itis meant that any one of the N (in a circular polynucleotide) or N−1 (ina linear polynucleotide) covalent linkages between residue positions ina single polynucleotide molecule are cleaved. Accordingly, in one vesselcontaining many copies of a polynucleotide, a single random break canoccur at different positions in different molecules.

[0041] As used herein, “substantially pure” means an object species isthe predominant species present (i.e., on a molar basis it is moreabundant than any other individual macromolecular species in thecomposition), and preferably a substantially purified fraction is acomposition wherein the object species comprises at least about 50percent (on a molar basis) of all macromolecular species present.Generally, a substantially pure composition will comprise more thanabout 80 to 90 percent of all macromolecular species present in thecomposition. Most preferably, the object species is purified toessential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species.

[0042] The term “homologous” means that one single-stranded nucleic acidsequence may hybridize to a complementary single-stranded nucleic acidsequence. The degree of hybridization may depend on a number of factorsincluding the amount of identity between the sequences and thehybridization conditions such as temperature and salt concentration asdiscussed later. Preferably the region of identity is greater than about5 bp, more preferably the region of identity is greater than 10 bp.Thus, “homologs” are nucleic acid molecules that are not identical butare capable of hybridizing to one another under physiologicalconditions. Double-stranded homologs are capable of hybridizing to oneanother following denaturation.

[0043] The term “heterologous” means that one single-stranded nucleicacid sequence is unable to hybridize to another single-stranded nucleicacid sequence or its complement. Thus areas of heterology means thatnucleic acid fragments or polynucleotides have areas or regions in thesequence which are unable to hybridize to another nucleic acid orpolynucleotide. Such regions are, for example, regions that are mutated.

[0044] The phrase “selectively (or specifically) hybridizes to” refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA).

[0045] The phrase “stringent hybridization conditions” refers toconditions under which a probe will hybridize to its target subsequence,typically in a complex mixture of nucleic acid, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, optionally 10 times background hybridization. Exemplarystringent hybridization conditions can be as following: 50% formamide,5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubatingat 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes canbe performed for 5, 15, 30, 60, 120, or more minutes. For PCR, atemperature of about 36° C. is typical for low stringency amplification,although annealing temperatures may vary between about 32° C. and 48° C.depending on primer length. For high stringency PCR amplification, atemperature of about 62° C. is typical, although high stringencyannealing temperatures can range from about 50° C. to about 65° C.,depending on the primer length and specificity. Typical cycle conditionsfor both high and low stringency amplifications include a denaturationphase of 90° C.-95° C. for 30 sec-2 min., an annealing phase lasting 30sec.-2 min., and an extension phase of about 72° C. for 1-2 min.

[0046] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

[0047] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues or nucleotides that are the same (i.e., about 60%identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher identity over a specified region whencompared and aligned for maximum correspondence over a comparison windowor designated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to, or may beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, or 150 or more in length.

[0048] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

[0049] A “comparison window”, as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local alignmentalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by theglobal alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)). Typically, the Smith & Waterman alignment with the defaultparameters are used for the purposes of this invention.

[0050] Another example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, typically with thedefault parameters, to determine percent sequence identity for thenucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

[0051] The term “amplification” means that the number of copies of anucleic acid sequence is increased.

[0052] The term “wild-type” means that the nucleic acid fragment doesnot comprise any mutations. As used herein, the term “wild type” isequivalent to “parental sequence”, i.e., a starting or referencesequence prior to the manipulation of the sequence. For example, amutation in the origin of the ColE1 plasmid has long been known toincrease the copy number of the plasmid (up to 300 per cell). See, e.g,Muesing et al., Cell, 24:235-242, 1981. This origin can be considered tobe a wild-type origin in the context of this invention.

[0053] The term “chimeric polynucleotide” means that the polynucleotidecomprises nucleotide regions which are wild-type and regions that aremutated. It may also mean that the polynucleotide comprises wild-typeregions from one polynucleotide and wild-type regions from anotherrelated polynucleotide.

[0054] The term “population” as used herein means a collection ofcomponents such as polynucleotides, nucleic acid fragments or proteins.A “mixed population” means a collection of components which belong tothe same family of nucleic acids or proteins (i.e. are related) butwhich differ in their sequence (i.e. are not identical) and hence intheir biological activity. A “library” necessarily implies a populationwherein at least two of the components is different in some aspect(chemical composition, length, etc.).

[0055] The term “specific nucleic acid fragment” means a nucleic acidfragment having certain end points and having a certain nucleic acidsequence. Two nucleic acid fragments wherein one nucleic acid fragmenthas the identical sequence as a portion of the second nucleic acidfragment but different ends comprise two different specific nucleic acidfragments. Two nucleic acid fragments with identical sequences butdifferent 5′ or 3′ ends comprise two different specific nucleic acidfragments.

[0056] The term “mutations” as used herein refers to changes in thesequence of a parental nucleic acid sequence. Mutations may be pointmutations such as transitions or transversions, or deletion orinsertions.

[0057] In the polynucleotide notation used herein, unless specifiedotherwise, the left-hand end of single-stranded polynucleotide sequencesis the 5′ end; the left-hand direction of double-stranded polynucleotidesequences is referred to as the 5′ direction. The direction of 5′ to 3′addition of nascent RNA transcripts is referred to as the transcriptiondirection; sequence regions on the DNA strand having the same sequenceas the RNA and which are 5′ to the 5′ end of the RNA transcript arereferred to as “upstream sequences”; sequence regions on the DNA strandhaving the same sequence as the RNA and which are 3′ to the 3′ end ofthe coding RNA transcript are referred to as “downstream sequences”.

[0058] As used herein the term “physiological conditions” refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable cultured yeast cell ormammalian cell. For example, the intracellular conditions in a yeastcell grown under typical laboratory culture conditions are physiologicalconditions. Suitable in vitro reaction conditions for in vitrotranscription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 mM NaCl orKCl, pH 6.5-8.5, 20-45° C. and 0.001-10 mM divalent cation (e.g., Mg⁺⁺,Ca⁺⁺); preferably about 150 mM NaCl or KCI, pH 7.2-7.6, 5 mM divalentcation, and often include 0.01-1.0 percent nonspecific protein (e.g.,BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) can often bepresent, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mMTris HCl, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or nonionic detergents and/or membrane fractionsand/or antifoam agents and/or scintillants.

[0059] As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

[0060] Introduction

[0061] Copy number is a genetic characteristic of each plasmid. Forexample, in the ColE1-type plasmids (such as plasmids of the familiespBR, pUC, and the like), the copy number is under the control of a DNAregion corresponding to the replication origin of the plasmid (ORI)[Peden, et.al. Gene, 22 (1983) 277-280]). A portion of this region,situated between bases 2970 and 3089 is transcribed into RNAs calledRNAI and RNAII. RNAI, in particular, is known to play a role in theregulation of the plasmid copy number. A schematic of a ColE1-relatedorigin of replication is shown if FIG. 1. FIG. 2 shows the sequence of aColE1-related origin of replication from the pBluescript plasmid. FIG. 3shows the DNA sequence encoding RNAII from a ColE1-type plasmid. Thepresent invention provides for mutations, often insertions or deletions,in the RNAI region of ColE1-related origins of replication that increasethe copy number of plasmids that harbor them, or alter the compatibilityof such plasmids.

[0062] Despite the recent advances in understanding the regulation ofColE1-type plasmids by RNAI, RNAII and specific enzyme activities,relatively few gain of function alterations in the origin of replicationare known that stably increase the copy number. Boros et al. [Gene, 30,(1984) 257-260] describe a mutant plasmid derived from pBR322. The copynumber of this plasmid per cell is increased by about 200-fold relativeto the copy number of pBR322. This increase in the number of copiesresults from a G to T transversion localized in position 3075 on the2846-3363 HinfI fragment, close to the 3′ end of the sequencetranscribed into RNAI. Muesing et al. [Cell, 24 (1981) 235-242] hadearlier demonstrated the same mutation in the plasmid ColE1(whosereplication origin is similar through the sequence to that of pBR322),also with an increase in the copy number of the said plasmid (up to 300per cell). Also, a high copy variant of pRSF1030 with a singlenucleotide change in the region encoding RNAI has recently beendescribed [Phillips, et al. Biotechniques 28 (2000) 400-408]. Thus, afew base pair changes have been described which increase the copy numberof low to medium copy number plasmids, however increases in copy numberat a magnitude of 3 fold or greater due to an insertion or deletion oralterations in compatibility have not been demonstrated to date.

[0063] Replication Origins

[0064] For a review of plasmid origin of replication families, see,e.g., del Solar, et.al. in Microbiology and Molecular Biology Reviews,62 (1998) 434-464. Origins of replication include ColE1 family originsas well as others that are distinct from ColE1. Those that do not belongto the ColE1-related family include those derived from the plasmids R1,R6K, pSC101, or pPS10.

[0065] ColE1-type origins of replication are common in plasmidsfrequently used in recombinant techniques. Examples include the pBRorigin and the ColE1-type origin of replication sequence comprisingresidues 1158 to 1825 of pBluescript. These residues are set forth inFIG. 2 and SEQ ID NO:1.

[0066] Mutations, e.g., substitutions, deletions, and/or insertions maybe introduced into the origin or replication using a number of methods.Current methods in widespread use for creating mutant sequences, forexample in a library format, are error-prone polymerase chain reaction(Caldwell & Joyce, (1992); Gram et al., Proc Natl Acad Sci 89:3576-80,1992) and cassette mutagenesis Arkin & Youvan, Proc Natl Acad Sci89:7811-5, 1992; Hermes et al., Proc Natl Acad Sci 87:696-700, 1990;Oliphant et al., Gene 44: 177-83 (1986); Stemmer & Morris, Biotechniques13: 214-20 (1992)], in which the specific region to be optimized isreplaced with a synthetically mutagenized oligonucleotide.Alternatively, mutator strains of host cells have been employed to addmutational frequency (Greener et al., Mol Biotechnol 7:189-95, 1997). Ineach case, a ‘mutant cloud’ Kauffman, (199) is generated around certainsites in the original sequence.

[0067] Methods of saturation mutagenesis utilizing random or partiallydegenerate primers that incorporate restriction sites have also beendescribed (Hill et al., Methods Enzymol 155:558-68; 1987; Oliphant etal., Gene 44:177-83; 1986; Reidhaar-Olson et al., Methods Enzymol208:564-86, 1991). A protocol has also been developed by which synthesisof an oligonucleotide is “doped” with non-native phosphoramidites,resulting in randomization of the gene section targeted for randommutagenesis Wang & Hoover, J Bacteriol 179:5812-9, 1997). This methodallows control of position selection, while retaining a randomsubstitution rate. Zaccolo & Gherardi, (J Mol Biol 285:775-83, 1999)describe a method of random mutagenesis utilizing pyrimidine and purinenucleoside analogs. U.S. Pat. No. 5,798,208 describes a “walk through”method, wherein a predetermined amino acid is introduced into a targetedsequence at pre-selected positions.

[0068] Methods for mutating a target gene by insertion and/or deletionmutations have also been developed. It has been demonstrated thatinsertion mutations could be accommodated in the interior ofstaphylococcal nuclease Keefe et al., Protein Sci 3:391-401, 1994).Examples of deletional mutagenesis methods developed include theutilization of an exonuclease (such as exonuclease III or Bal31) orthrough oligonucleotide directed deletions incorporating point deletionsNer et al., Nucleic Acids Res 17:4015-23, 1989). Additionally, Lietzdescribes a method whereby oligonucleotides with random sequences may becombined with PCR to induce insertions and deletions. Enhancement offunction by this technique has not been shown, and the capacity toovermutagenize (i.e., make too many insertions or deletions perpolynucleotide) is substantial in this method (see, e.g., U.S. Pat. No.6,251,604.

[0069] A technique often used to evolve proteins in vitro is known as“DNA Shuffling”. In this method, a library of gene modifications iscreated by fragmenting homologous sequences of a gene, allowing thefragments to randomly anneal to one another, and filling in theoverhangs with polymerase. A full length gene library is thenreconstructed with polymerase chain reaction (PCR). The utility of thismethod occurs at the step of annealing, whereby homologous sequences mayanneal to one another, producing sequences with attributes of bothstarting sequences. In effect, the method affects recombination betweentwo or more genes that are homologous, but that contain significantdifferences at several positions. It has been shown that creation of thelibrary using several homologous sequences allows a sampling of moresequence space than using a randomly mutated single starting sequence(see, e.g., Crameri et al., Nature 391:288-91, 1998). This effect islikely due to the fact that years of evolution have already selected fordifferent advantageous or neutral mutations amongst the homologs of thedifferent species. Starting with homologs, then, appreciably limits thenumber of deleterious mutations in the creation of the library which isto be screened. Combinatorially rearranging the advantageous positionsof the homologs can apparently allow for an optimized secondary proteinstructure for catalyzing a biochemical reaction. The resulting evolvedprotein appears to contain positive features contributed from each ofthe starting sequences, which results in drastically improved functionfollowing selection.

[0070] A recently described technology describes the ability to makedeletions or additions to random positions within a circularpolynucleotide (see, e.g, WO0216642). This technology is especiallysuited to the application of producing high-copy variants of ColE1-typeplasmids due to the known importance of RNA secondary structure inreplication initiation. Insertions or deletions of varying length can bemade at any position in the origin of replication, and a screen for highcopy-number can be done to identify useful mutants.

[0071] Position of Mutations in the Origin of Replication

[0072] Origins of replication of the invention that allow circular DNAmolecules to replicate to a high copy number or that confercompatibility on a plasmid can be generated by mutating, e.g.,inserting, substituting, or deleting, residues at a position between,and including, position 1 to 210, as determined with reference to SEQ IDNO:1. Often, high copy number and/or compatibility origins of theinvention comprise mutations, relative to SEQ ID NO:1, within the regioncorresponding to SEQ ID NO:1 from position 1 to 150. For example,origins of replication of the invention can have deletions of one ormore nucleotides at a position in the region of SEQ ID NO:1 fromposition 1 to 150. Such deletions can occur at any position.Additionally, deletions can be at more than one position. The deletionsvary in length. Deletions are usually less than 100 residues, often lessthan 50 residues, and most often less than 25, 20, or 15 residues, e.g.,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residues.

[0073] Similarly, insertions in origins can occur at any of positions 1to 210 of SEQ ID NO:1. Typically, insertions are in positions 1 to 150of SEQ ID NO:1. Such insertions can be at any position. Further,multiple insertions can be present in the origins of the invention. Theinsertions vary in length. For example, the insertions are usually lessthan 100 base pairs, often less than 50 base pairs, and most often lessthan 25, 20, or 15 residues, e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,or 1.

[0074] Substitutions may also be introduced into the region comprisingpositions 1 to 210, of SEQ ID NO:1, typically positions 1 to 150 of SEQID NO:1. Typically, more than one position is substituted. For example,usually less than 100 positions are substituted, often less than 50positions, and most often less than 25, 20, or 15 positions, e.g., 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0075] Additionally, combinations of substitutions, insertions, ordeletions as described above can be present in a ColE1-type plasmid ofthe invention. For example, an origin of the invention can comprise botha deletion and substitutions at other positions relative to SEQ ID NO:1,e.g, ori mutant 4.1.

[0076] In preferred embodiments, the mutations in the origin occur inthe region encoding RNAI (identified in FIGS. 2 and 4), or within 10nucleotides of the region encoding RNAI. Often, mutations occur withinthe region from position 39 to position 66 of SEQ ID NO:1, or positions91 through 135 of SEQ ID NO:1. FIG. 6 shows the structure of the RNA IIregion of Bluescript that includes the RNAI sequences. The positions ofthe ori mutations described below are shown on the structure, except forori 2.2, where the deletion occurs outside of the RNAI region.

[0077] The invention includes, but is not limited to, the followingembodiments. The positions of the residues are indicated with referenceto SEQ ID NO:1.

[0078] (a) Origin mutant 3.1 (SEQ ID NO:3) comprises a deletion of 168residues, 68 of which are at the 5′ end of the origin of replication andinclude the 5′ 30 positions of the DNA encoding RNAII. The other 100residues of the deletion are outside of the reference origin as shown inSEQ ID NO:1.

[0079] (b) Origin mutant 3.2 (SEQ ID NO:4) comprises a 4 nucleotide GCTAdeletion from positions 57 to 60 in the origin of replication, whichcorresponds to positions 18 to 21 of the RNAII transcript.

[0080] (c) Origin mutant 3.3 (SEQ ID NO:5) comprises a 3 nucleotide GCAdeletion from position 125 to 127 of the origin, which corresponds toposition 86 to 88 of RNAII. This can also be considered to be a 3nucleotide deletion of CAG at positions 126 to 128, as this results inthe same sequence in ori 3.3.

[0081] (d) Origin mutant 3.4 (SEQ ID NO:6) comprises an 11 nucleotideCAAACAAAAAA deletion from positions 40 to 50 of the origin, whichcorresponds to positions 2 to 12 of RNAII.

[0082] (e) Origin mutant 2.1 (SEQ ID NO:7) has a 6 base deletion fromnucleotides 59-64 (relative to pBluescript.

[0083] (f) Origin mutant 2.2 (SEQ ID NO:8) has a 3 base deletion from192-194.

[0084] (g) Origin mutant 2.3 (SEQ ID NO:9) has a 7 base deletion from128-134.

[0085] (h) Origin mutant 4.1 (SEQ ID NO:10) has a two base deletion at61-62, but also has two nucleotides altered near the deletion site (C58Gand A60G).

[0086] (i) Origin mutant 5.1 (SEQ ID NO:11) has an 11 base deletion from93-103.

[0087] (j) Origin mutant 5.2 (SEQ ID NO:12) has a 5 base deletion from47-51.

[0088] (k) Origin mutant 5.3 (SEQ ID NO:13) has a 7 base deletion from59-65.

[0089] Alignments of the ori 5′ regions of SEQ ID NOs:4-13 with thecorresponding region of pBluescript are shown in FIG. 4.

[0090] Methods to prepare the polynucleotides comprising high-copynumber origins of replication of the present invention are well known inthe art. Plasmids containing expected high-copy number origins ofreplication can be readily assayed as described below to determine theparticular plasmid's copy number. The production of high-copy plasmidscould be accomplished through various mutagenesis process, e.g.,site-directed mutagenesis. See, for example, Sambrook & Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 2001, 3rdedition “Oligonucleotide-Mediated Mutagenesis,” which is incorporatedherein by reference. Site-directed mutagenesis is generally accomplishedby site-specific primer-directed mutagenesis. This technique is nowstandard in the art and is conducted using a synthetic oligonucleotideprimer complementary to a single-stranded phage DNA to be mutagenizedexcept for a limited deletion or insertion representing the desiredmutation. Briefly, the synthetic oligonucleotide is used as a primer todirect synthesis of a strand complementary to the plasmid or phage, andthe resulting double-stranded DNA is transformed into a phage-supportinghost bacterium. The resulting bacteria can be assayed by, for example,DNA sequence analysis or probe hybridization to identify those plaquescarrying the desired mutated gene sequence. Alternatively, “recombinantPCR” methods can be employed [Innis et al. editors, PCR Protocols, SanDiego, Academic Press, 1990, Chapter 22, Entitled “Recombinant PCR”,Higuchi, pages 177-183].

[0091] Use of the present invention typically would involve theconstruction of a circular polynucleotide comprising a high-copy originof replication as described herein. Techniques for the construction ofsuch recombinant DNA molecules are well known in the art, as described,for example, in Sambrook and Russell, eds, Molecular Cloning: ALaboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor LaboratoryPress, 2001; and Current Protocols in Molecular Biology, Ausubel, ed.John Wiley & Sons, Inc. New York (1997). Such a high-copy plasmid mayalso comprise a gene of interest, which is to be expressed in a hostcell. The gene of interest may be determined by the individual interestof the investigator. Such a gene may encode a pharmaceutical, anindustrial enzyme, or any other RNA or protein molecule. The high-copyplasmid is preferably inserted into a host cell, preferably aprokaryote. Methods for inserting genes into prokaryotes includeelectroporation, heat shock transformation, and phage transduction.

[0092] The host strains suitable for the multiplication of the plasmidsconforming to the invention and to the expression of the genes carriedby these plasmids are the same as those which permit the multiplicationof the corresponding wild type plasmids and the expression of the geneswhich they carry, and the behaviour and the growth of the strainstransformed by the plasmids conforming to the invention are identical tothose of the strains carrying the wild type plasmids.

[0093] The subject of the present invention is in addition a process forthe multiplication of the plasmids conforming to the invention, whichprocess is characterized in that, in a first step, an appropriate hostbacterial strain is transformed with at least one of the said plasmids,and in a second step, the said bacterial strain is cultured.

[0094] The invention also encompasses:

[0095] A process for the amplification of a DNA sequence, which processis characterized in that, in a first step, the said sequence is insertedin a plasmid conforming to the invention, and in that, in a second step,the multiplication of the said plasmid is carried out as indicatedabove.

[0096] A process for the production of polypeptides by geneticengineering, which process is characterized in that, in a first step,the gene encoding the said polypeptide is inserted in a plasmidconforming to the invention, in a second step, an appropriate hostbacterial strain is transformed with the said plasmid, and in a thirdstep, the said bacterial strain is cultured under conditions appropriatefor the expression of the said gene.

[0097] Determination of Plasmid Copy Number

[0098] Relative Copy Number

[0099] One method to determine the relative copy number of plasmids isdescribed in U.S. Pat. No. 4,703,012. In this method, plasmids of anormal copy number are cultured in parallel with a test high-copyplasmid. The bacteria are lysed, and the plasmids are compared byagarose gel electrophoresis at various dilutions. If the test plasmidstains more intensely with ethidium bromide at a given dilution comparedto the normal copy plasmid, then its copy number is increased by anamount proportional to the increase in staining. The plasmid of normalcopy number may be any ColE1-related plasmid. Examples of suchColE1-related plasmids are pMB1, pBR322, the pUC series, p15A, and thepBluescript series. A test plasmid may be any ColE1-related plasmid thatis suspected of having a high copy number.

[0100] Alternatively, plasmid copy number can be determined as aproportion of chromosome copies. In this method, bacterial cells arelysed, protein is digested by a protease, and total DNA is analyzed byagarose gel electrophoresis. The relative amounts of plasmid tochromosomal DNA can be determined for a normal copy plasmid, andcompared to a test high-copy plasmid. This comparison can be quantifiedusing ethidium bromide staining of said agarose gels. Normal copyplasmids could be plasmids harboring ColE1-related origins ofreplication such as pMB1, pBR322, the pUC series, p15A, and thepBluescript series.

[0101] Absolute Copy Number

[0102] The absolute copy number of a plasmid within a cell can bedetermined by analyzing the average number of plasmid molecules within acell in a given culture. In this method, a culture of cells is growncontaining the test plasmid, and an aliquot of cells are lysed in midlog phase. Plasmid DNA is prepared from this aliquot by any of severalstandard techniques. The plasmid DNA concentration, and absolute amount,are determined by spectroscopy or fluorometry. The remaining cells arethen plated in multiple dilutions on LB plates with the appropriateantibiotic selection. The colonies growing on these plates are thencounted to give an accurate measure of the viable cells in the originalculture. The copy number is then determined by deducing the number ofcopies/viable cell using the data acquired in the aforementionedprocess. Alternatively, the optical density of a bacterial colony oftenrelates linearly to its cell count. Hence, optical density can be usedas the denominator of the preceding calculation.

[0103] Plasmid Compatibility Testing

[0104] Compatibility of plasmids may be tested by inserting two or moreplasmids into the same bacterial cell. The plasmids should preferablyhave some distinguishing characteristic between them. The distinguishingcharacteristic may be resistance to an antibiotic, as is well known inthe art. For example, one plasmid may confer resistance to ampicillin byharboring a beta-lactamase gene, whereas the second plasmid may conferresistance to a different antibiotic, such as tetracycline orchloramphenicol. When the bacterial cells are selected in the presenceof both antibiotics, all of the cells in that population will harborboth plasmids. When one of the antibiotics is removed, cells withcompatible plasmids will retain the second plasmid, whereas incompatibleplasmids will lose the plasmid that is not under selection pressure.Cells retaining the second plasmid can be identified by replica platingthe cells from plates containing the first antibiotic onto new agarplates which contain the second antibiotic by techniques well known tothose skilled in the art.

EXAMPLES

[0105] The following examples are provided by way of illustration onlyand not by way of limitation. Those of skill in the art will readilyrecognize a variety of noncritical parameters that could be changed ormodified to yield essentially similar results.

[0106] Four plasmids derived from the ColE1-related pBluescript wereconstructed according to known methodology (see, e.g., WO0216642). Theseplasmids were identified based on their increased expression ofbeta-galactosidase, which requires the alpha peptide encoded by theplasmids.

[0107] Beta-galactosidase expression was evaluated by plating theTOP10F′ bacteria on LB agar plates containing the colorimetric substrateX-Gal, either with or without the inducer of lacZ IPTG. The four originof replication mutants showed increased blue colony color compared tothe wild-type pBluescript plasmid, both in the presence and in theabsence of the inducer IPTG. The number of copies per viable cell wasdetermined by growing 100 ml cultures of DH10B e.coli containing each ofthe plasmids, preparing the plasmids from a 50 ml aliquot by QIAGENcolumns (QIAGEN, Chatsworth, Calif.), determining the absolute amount ofplasmid DNA in the preparation by O.D.₂₆₀, and plating dilutions of theremaining 50 ml of culture in order to determine the number of viablecells in the original culture.

[0108] An exemplary experiment shows that the copy number per viablecell was increased nearly ten fold compared to wild-type plasmids (FIG.5). The plasmids were sequenced and the only alterations from wild-typewere in the origin of replication. The sequences of the origins ofreplication are set forth in SEQ ID NOs:3-6.

[0109] Additional plasmids were also constructed as above. Sequences ofhigh copy number plasmids identified as set forth herein are shown inSEQ ID NOs:7-14.

[0110] Of these sequences, ori 3.3 and ori 5.1 are compatibilitymutants, i.e., these plasmids can co-exist with ColE1-related origins,e.g., wild-type ColE1origins, in a single cell.

[0111] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

[0112] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Table of Sequences SEQ I.DNO: 1 ColE1-related origin from pBluescript (residues 1158-1825)TCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACAT GTTCTTTCCTGCGTTAT SEQID NO: 2 DNA encoding RNAII from the ColE1-related plasmid pBluescriptGCAAACAAAAAAACC ACCGCTACCAGCGGT GGTTTGTTTGCCGGA TCAAGAGCTACCAACTCTTTTTCCGAAGGT AACTGGCTTCAGCAG AGCGCAGATACCAAA TACTGTCCTTCTAGTGTAGCCGTAGTTAGG CCACCACTTCAAGAA CTCTGTAGCACCGCC TACATACCTCGCTCTGCTAATCCTGTTACC AGTGGCTGCTGCCAG TGGCGATAAGTCGTG TCTTACCGGGTTGGACTCAAGACGATAGTT ACCGGATAAGGCGCA GCGGTCGGGCTGAAC GGGGGGTTCGTGCACACAGCCCAGCTTGGA GCGAACGACCTACAC CGAACTGAGATACCT ACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAA GGCGGACAGGTATCC GGTAAGCGGCAGGGTCGGAACAGGAGAGCG CACGAGGGAGCTTCC AGGGGGAAACGCCTG GTATCTTTATAGTCCTGTCGGGTTTCGCCA CCTCTGACTTGAGCG TCGATTTTTGTGATG CTCGTCAGGGGGGCGGAGCCTATGGAAA SEQ ID NO: 3 DNA encoding origin 3.1, a high-copy variantof a ColE1 plasmid. GGTTTGTTTGCCGGA TCAAGAGCTACCAAC TCTTTTTCCGAAGGTAACTGGCTTCAGCAG AGCGCAGATACCAAA TACTGTCCTTCTAGT GTAGCCGTAGTTAGGCCACCACTTCAAGAA CTCTGTAGCACCGCC TACATACCTCGCTCT GCTAATCCTGTTACCAGTGGCTGCTGCCAG TGGCGATAAGTCGTG TCTTACCGGGTTGGA CTCAAGACGATAGTTACCGGATAAGGCGCA GCGGTCGGGCTGAAC GGGGGGTTCGTGCAC ACAGCCCAGCTTGGAGCGAACGACCTACAC CGAACTGAGATACCT ACAGCGTGAGCTATG AGAAAGCGCCACGCTTCCCGAAGGGAGAAA GGCGGACAGGTATCC GGTAAGCGGCAGGGT CGGAACAGGAGAGCGCACGAGGGAGCTTCC AGGGGGAAACGCCTG GTATCTTTATAGTCC TGTCGGGTTTCGCCACCTCTGACTTGAGCG TCGATTTTTGTGATG CTCGTCAGGGGGGCG GAGCCTATGGAAAAACGCCAGCAACGCGGC CTTTTTACGGTTCCT GGCCTTTTGCTGGCC TTTTGCTCACATGTTCTTTCCTGCGTTAT SEQ ID NO: 4 DNA encoding origin 3.2, a high-copy variantof a ColE1 plasmid. TCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCCCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTC TTTCCTGCGTTAT SEQ IDNO: 5 DNA encoding origin 3.3, a high-copy variant of a ColE1 plasmid.TCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT CTTTCCTGCGTTAT SEQ IDNO: 6 DNA encoding origin 3.4, a high-copy variant of a ColE1 plasmidTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTG CGTTAT SEQ ID NO: 7DNA encoding ori 2.1, a high-copy variant of a ColE1 plasmidTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT TCCTGCGTTAT SEQ IDNO: 8 DNA encoding ori 2.2, a high-copy variant of a ColE1 plasmidTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGNTCCTGGNCNTTTGCTGGCCTTTTGCTCACATGTT CTTTCCTGCGTTAT SEQ IDNO: 9 DNA encoding ori 2.3, a high-copy variant of a ColE1 plasmidTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTT CCTGCGTTAT SEQ ID NO:10 DNA encoding ori 4.1, a high-copy variant of a ColE1 plasmidTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGGTGACCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCANGGTCGGAACAGGAGAGCGCACGANGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT TCTTTCCTGCGTTAT SEQID NO: 11 DNA encoding ori 5.1, a high-copy variant of a ColE1 plasmidTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGNCTTTTNGCTCACATGTTCTTTCCT GCGTTAT SEQ ID NO: 12DNA encoding ori 5.2, a high-copy variant of a ColE1 plasmidTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT TTCCTGCGTTAT SEQ IDNO: 13 DNA encoding ori 5.3, a high-copy variant of a ColE1 plasmidTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTT CCTGCGTTAT

[0113]

1 27 1 667 DNA Artificial Sequence Description of Artificial SequenceColE1-related origin of replication (ORI) from pBluescript plasmid(residues 1158-1825) 1 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgcaaacaaaaaa accaccgcta 60 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactctttttccgaa ggtaactggc 120 ttcagcagag cgcagatacc aaatactgtc cttctagtgtagccgtagtt aggccaccac 180 ttcaagaact ctgtagcacc gcctacatac ctcgctctgctaatcctgtt accagtggct 240 gctgccagtg gcgataagtc gtgtcttacc gggttggactcaagacgata gttaccggat 300 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacacagcccagctt ggagcgaacg 360 acctacaccg aactgagata cctacagcgt gagctatgagaaagcgccac gcttcccgaa 420 gggagaaagg cggacaggta tccggtaagc ggcagggtcggaacaggaga gcgcacgagg 480 gagcttccag ggggaaacgc ctggtatctt tatagtcctgtcgggtttcg ccacctctga 540 cttgagcgtc gatttttgtg atgctcgtca ggggggcggagcctatggaa aaacgccagc 600 aacgcggcct ttttacggtt cctggccttt tgctggccttttgctcacat gttctttcct 660 gcgttat 667 2 553 DNA Artificial SequenceDescription of Artificial Sequence DNA encoding RNAII from ColE1-relatedorigin of replication (ORI) from pBluescript plasmid 2 gcaaacaaaaaaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 60 tctttttccgaaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 120 gtagccgtagttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 180 gctaatcctgttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 240 ctcaagacgatagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 300 acagcccagcttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 360 agaaagcgccacgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 420 cggaacaggagagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 480 tgtcgggtttcgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 540 gagcctatggaaa 553 3 599 DNA Artificial Sequence Description of Artificial SequenceDNA encoding origin of replication (ori5′) mutant 3.1 (ori 3.1), ahigh-copy variant of ColE1-type plasmid 3 ggtttgtttg ccggatcaagagctaccaac tctttttccg aaggtaactg gcttcagcag 60 agcgcagata ccaaatactgtccttctagt gtagccgtag ttaggccacc acttcaagaa 120 ctctgtagca ccgcctacatacctcgctct gctaatcctg ttaccagtgg ctgctgccag 180 tggcgataag tcgtgtcttaccgggttgga ctcaagacga tagttaccgg ataaggcgca 240 gcggtcgggc tgaacggggggttcgtgcac acagcccagc ttggagcgaa cgacctacac 300 cgaactgaga tacctacagcgtgagctatg agaaagcgcc acgcttcccg aagggagaaa 360 ggcggacagg tatccggtaagcggcagggt cggaacagga gagcgcacga gggagcttcc 420 agggggaaac gcctggtatctttatagtcc tgtcgggttt cgccacctct gacttgagcg 480 tcgatttttg tgatgctcgtcaggggggcg gagcctatgg aaaaacgcca gcaacgcggc 540 ctttttacgg ttcctggccttttgctggcc ttttgctcac atgttctttc ctgcgttat 599 4 663 DNA ArtificialSequence Description of Artificial Sequence DNA encoding origin ofreplication (ori5′) mutant 3.2 (ori 3.2), a high-copy variant ofColE1-type plasmid 4 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgcaaacaaaaaa accaccccag 60 cggtggtttg tttgccggat caagagctac caactctttttccgaaggta actggcttca 120 gcagagcgca gataccaaat actgtccttc tagtgtagccgtagttaggc caccacttca 180 agaactctgt agcaccgcct acatacctcg ctctgctaatcctgttacca gtggctgctg 240 ccagtggcga taagtcgtgt cttaccgggt tggactcaagacgatagtta ccggataagg 300 cgcagcggtc gggctgaacg gggggttcgt gcacacagcccagcttggag cgaacgacct 360 acaccgaact gagataccta cagcgtgagc tatgagaaagcgccacgctt cccgaaggga 420 gaaaggcgga caggtatccg gtaagcggca gggtcggaacaggagagcgc acgagggagc 480 ttccaggggg aaacgcctgg tatctttata gtcctgtcgggtttcgccac ctctgacttg 540 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcctatggaaaaac gccagcaacg 600 cggccttttt acggttcctg gccttttgct ggccttttgctcacatgttc tttcctgcgt 660 tat 663 5 664 DNA Artificial SequenceDescription of Artificial Sequence DNA encoding origin of replication(ori5′) mutant 3.3 (ori 3.3), a high-copy variant of ColE1-type plasmid5 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 60ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 120ttcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 180aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 240gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 300gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 360tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 420agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 480cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 540gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 600gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 660ttat 664 6 656 DNA Artificial Sequence Description of ArtificialSequence DNA encoding origin of replication (ori5′) mutant 3.4 (ori3.4), a high-copy variant of ColE1-type plasmid 6 tcttgagatc ctttttttctgcgcgtaatc tgctgcttga ccaccgctac cagcggtggt 60 ttgtttgccg gatcaagagctaccaactct ttttccgaag gtaactggct tcagcagagc 120 gcagatacca aatactgtccttctagtgta gccgtagtta ggccaccact tcaagaactc 180 tgtagcaccg cctacatacctcgctctgct aatcctgtta ccagtggctg ctgccagtgg 240 cgataagtcg tgtcttaccgggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcgggctga acggggggttcgtgcacaca gcccagcttg gagcgaacga cctacaccga 360 actgagatac ctacagcgtgagctatgaga aagcgccacg cttcccgaag ggagaaaggc 420 ggacaggtat ccggtaagcggcagggtcgg aacaggagag cgcacgaggg agcttccagg 480 gggaaacgcc tggtatctttatagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 540 atttttgtga tgctcgtcaggggggcggag cctatggaaa aacgccagca acgcggcctt 600 tttacggttc ctggccttttgctggccttt tgctcacatg ttctttcctg cgttat 656 7 661 DNA ArtificialSequence Description of Artificial Sequence DNA encoding origin ofreplication (ori5′) mutant 2.1 (ori 2.1), a high-copy variant ofColE1-type plasmid 7 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgcaaacaaaaaa accaccggcg 60 gtggtttgtt tgccggatca agagctacca actctttttccgaaggtaac tggcttcagc 120 agagcgcaga taccaaatac tgttcttcta gtgtagccgtagttaggcca ccacttcaag 180 aactctgtag caccgcctac atacctcgct ctgctaatcctgttaccagt ggctgctgcc 240 agtggcgata agtcgtgtct taccgggttg gactcaagacgatagttacc ggataaggcg 300 cagcggtcgg gctgaacggg gggttcgtgc acacagcccagcttggagcg aacgacctac 360 accgaactga gatacctaca gcgtgagcta tgagaaagcgccacgcttcc cgaagggaga 420 aaggcggaca ggtatccggt aagcggcagg gtcggaacaggagagcgcac gagggagctt 480 ccagggggaa acgcctggta tctttatagt cctgtcgggtttcgccacct ctgacttgag 540 cgtcgatttt tgtgatgctc gtcagggggg cggagcctatggaaaaacgc cagcaacgcg 600 gcctttttac ggttcctggc cttttgctgg ccttttgctcacatgttctt tcctgcgtta 660 t 661 8 664 DNA Artificial SequenceDescription of Artificial Sequence DNA encoding origin of replication(ori5′) mutant 2.2 (ori 2.2), a high-copy variant of ColE1-type plasmid8 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 60ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 120ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac 180ttcaagaact cagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 240gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 300gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 360tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 420agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 480cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 540gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 600gcggcctttt tacggntcct ggncntttgc tggccttttg ctcacatgtt ctttcctgcg 660ttat 664 9 660 DNA Artificial Sequence Description of ArtificialSequence DNA encoding origin of replication (ori5′) mutant 2.3 (ori2.3), a high-copy variant of ColE1-type plasmid 9 tcttgagatc ctttttttctgcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 60 ccagcggtgg tttgtttgccggatcaagag ctaccaactc tttttccgaa ggtaactggc 120 ttcagcagat accaaatactgttcttctag tgtagccgta gttaggccac cacttcaaga 180 actctgtagc accgcctacatacctcgctc tgctaatcct gttaccagtg gctgctgcca 240 gtggcgataa gtcgtgtcttaccgggttgg actcaagacg atagttaccg gataaggcgc 300 agcggtcggg ctgaacggggggttcgtgca cacagcccag cttggagcga acgacctaca 360 ccgaactgag atacctacagcgtgagctat gagaaagcgc cacgcttccc gaagggagaa 420 aggcggacag gtatccggtaagcggcaggg tcggaacagg agagcgcacg agggagcttc 480 cagggggaaa cgcctggtatctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 540 gtcgattttt gtgatgctcgtcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 600 cctttttacg gttcctggccttttgctggc cttttgctca catgttcttt cctgcgttat 660 10 665 DNA ArtificialSequence Description of Artificial Sequence DNA encoding origin ofreplication (ori5′) mutant 4.1 (ori 4.1), a high-copy variant ofColE1-type plasmid 10 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgcaaacaaaaaa accaccggtg 60 accggtggtt tgtttgccgg atcaagagct accaactctttttccgaagg taactggctt 120 cagcagagcg cagataccaa atactgttct tctagtgtagccgtagttag gccaccactt 180 caagaactct gtagcaccgc ctacatacct cgctctgctaatcctgttac cagtggctgc 240 tgccagtggc gataagtcgt gtcttaccgg gttggactcaagacgatagt taccggataa 300 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacagcccagcttgg agcgaacgac 360 ctacaccgaa ctgagatacc tacagcgtga gctatgagaaagcgccacgc ttcccgaagg 420 gagaaaggcg gacaggtatc cggtaagcgg canggtcggaacaggagagc gcacgangga 480 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtcgggtttcgcc acctctgact 540 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagcctatggaaaa acgccagcaa 600 cgcggccttt ttacggttcc tggccttttg ctggccttttgctcacatgt tctttcctgc 660 gttat 665 11 657 DNA Artificial SequenceDescription of Artificial Sequence DNA encoding origin of replication(ori5′) mutant 5.1 (ori 5.1), a high-copy variant of ColE1-type plasmid11 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 60ccagcggtgg tttgtttgcc ggatcaagag ctttccgaag gtaactggct tcagcagagc 120gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 180tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 240cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 360actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 420ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 480gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 540atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 600tttacggttc ctggcctttt gctggncttt tngctcacat gttctttcct gcgttat 657 12662 DNA Artificial Sequence Description of Artificial Sequence DNAencoding origin of replication (ori5′) mutant 5.2 (ori 5.2), a high-copyvariant of ColE1-type plasmid 12 tcttgagatc ctttttttct gcgcgtaatctgctgcttgc aaacaaccac cgctaccagc 60 ggtggtttgt ttgccggatc aagagctaccaactcttttt ccgaaggtaa ctggcttcag 120 cagagcgcag ataccaaata ctgttcttctagtgtagccg tagttaggcc accacttcaa 180 gaactctgta gcaccgccta catacctcgctctgctaatc ctgttaccag tggctgctgc 240 cagtggcgat aagtcgtgtc ttaccgggttggactcaaga cgatagttac cggataaggc 300 gcagcggtcg ggctgaacgg ggggttcgtgcacacagccc agcttggagc gaacgaccta 360 caccgaactg agatacctac agcgtgagctatgagaaagc gccacgcttc ccgaagggag 420 aaaggcggac aggtatccgg taagcggcagggtcggaaca ggagagcgca cgagggagct 480 tccaggggga aacgcctggt atctttatagtcctgtcggg tttcgccacc tctgacttga 540 gcgtcgattt ttgtgatgct cgtcaggggggcggagccta tggaaaaacg ccagcaacgc 600 ggccttttta cggttcctgg ccttttgctggccttttgct cacatgttct ttcctgcgtt 660 at 662 13 660 DNA ArtificialSequence Description of Artificial Sequence DNA encoding origin ofreplication (ori5′) mutant 5.3 (ori 5.3), a high-copy variant ofColE1-type plasmid 13 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgcaaacaaaaaa accaccgcgg 60 tggtttgttt gccggatcaa gagctaccaa ctctttttccgaaggtaact ggcttcagca 120 gagcgcagat accaaatact gttcttctag tgtagccgtagttaggccac cacttcaaga 180 actctgtagc accgcctaca tacctcgctc tgctaatcctgttaccagtg gctgctgcca 240 gtggcgataa gtcgtgtctt accgggttgg actcaagacgatagttaccg gataaggcgc 300 agcggtcggg ctgaacgggg ggttcgtgca cacagcccagcttggagcga acgacctaca 360 ccgaactgag atacctacag cgtgagctat gagaaagcgccacgcttccc gaagggagaa 420 aggcggacag gtatccggta agcggcaggg tcggaacaggagagcgcacg agggagcttc 480 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtttcgccacctc tgacttgagc 540 gtcgattttt gtgatgctcg tcaggggggc ggagcctatggaaaaacgcc agcaacgcgg 600 cctttttacg gttcctggcc ttttgctggc cttttgctcacatgttcttt cctgcgttat 660 14 270 DNA Artificial Sequence Description ofArtificial Sequence DNA encoding origin of replication (ori5′) mutantpBS, a high-copy variant of ColE1-type plasmid 14 tcttgagatc ctttttttctgcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 60 ccagcggtgg tttgtttgccggatcaagag ctaccaactc tttttccgaa ggtaactggc 120 ttcagcagag cgcagataccaaatactgtt cttctagtgt agccgtagtt aggccaccac 180 ttcaagaact ctgtagcaccgcctacatac ctcgctctgc taatcctgtt accagtggct 240 gctgccagtg gcgataagtcgtgtcttacc 270 15 264 DNA Artificial Sequence Description of ArtificialSequence DNA encoding origin of replication (ori5′) mutant 2.1 (ori2.1), a high-copy variant of ColE1-type plasmid 15 tcttgagatc ctttttttctgcgcgtaatc tgctgcttgc aaacaaaaaa accaccggcg 60 gtggtttgtt tgccggatcaagagctacca actctttttc cgaaggtaac tggcttcagc 120 agagcgcaga taccaaatactgttcttcta gtgtagccgt agttaggcca ccacttcaag 180 aactctgtag caccgcctacatacctcgct ctgctaatcc tgttaccagt ggctgctgcc 240 agtggcgata agtcgtgtcttacc 264 16 267 DNA Artificial Sequence Description of ArtificialSequence DNA encoding origin of replication (ori5′) mutant 2.2 (ori2.2), a high-copy variant of ColE1-type plasmid 16 tcttgagatc ctttttttctgcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 60 ccagcggtgg tttgtttgccggatcaagag ctaccaactc tttttccgaa ggtaactggc 120 ttcagcagag cgcagataccaaatactgtt cttctagtgt agccgtagtt aggccaccac 180 ttcaagaact cagcaccgcctacatacctc gctctgctaa tcctgttacc agtggctgct 240 gccagtggcg ataagtcgtgtcttacc 267 17 263 DNA Artificial Sequence Description of ArtificialSequence DNA encoding origin of replication (ori5′) mutant 2.3 (ori2.3), a high-copy variant of ColE1-type plasmid 17 tcttgagatc ctttttttctgcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 60 ccagcggtgg tttgtttgccggatcaagag ctaccaactc tttttccgaa ggtaactggc 120 ttcagcagat accaaatactgttcttctag tgtagccgta gttaggccac cacttcaaga 180 actctgtagc accgcctacatacctcgctc tgctaatcct gttaccagtg gctgctgcca 240 gtggcgataa gtcgtgtcttacc 263 18 266 DNA Artificial Sequence Description of ArtificialSequence DNA encoding origin of replication (ori5′) mutant 3.2 (ori3.2), a high-copy variant of ColE1-type plasmid 18 tcttgagatc ctttttttctgcgcgtaatc tgctgcttgc aaacaaaaaa accaccccag 60 cggtggtttg tttgccggatcaagagctac caactctttt tccgaaggta actggcttca 120 gcagagcgca gataccaaatactgttcttc tagtgtagcc gtagttaggc caccacttca 180 agaactctgt agcaccgcctacatacctcg ctctgctaat cctgttacca gtggctgctg 240 ccagtggcga taagtcgtgtcttacc 266 19 267 DNA Artificial Sequence Description of ArtificialSequence DNA encoding origin of replication (ori5′) mutant 3.3 (ori3.3), a high-copy variant of ColE1-type plasmid 19 tcttgagatc ctttttttctgcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 60 ccagcggtgg tttgtttgccggatcaagag ctaccaactc tttttccgaa ggtaactggc 120 ttcagagcgc agataccaaatactgttctt ctagtgtagc cgtagttagg ccaccacttc 180 aagaactctg tagcaccgcctacatacctc gctctgctaa tcctgttacc agtggctgct 240 gccagtggcg ataagtcgtgtcttacc 267 20 259 DNA Artificial Sequence Description of ArtificialSequence DNA encoding origin of replication (ori5′) mutant 3.4 (ori3.4), a high-copy variant of ColE1-type plasmid 20 tcttgagatc ctttttttctgcgcgtaatc tgctgcttga ccaccgctac cagcggtggt 60 ttgtttgccg gatcaagagctaccaactct ttttccgaag gtaactggct tcagcagagc 120 gcagatacca aatactgttcttctagtgta gccgtagtta ggccaccact tcaagaactc 180 tgtagcaccg cctacatacctcgctctgct aatcctgtta ccagtggctg ctgccagtgg 240 cgataagtcg tgtcttacc 25921 268 DNA Artificial Sequence Description of Artificial Sequence DNAencoding origin of replication (ori5′) mutant 4.1 (ori 4.1), a high-copyvariant of ColE1-type plasmid 21 tcttgagatc ctttttttct gcgcgtaatctgctgcttgc aaacaaaaaa accaccggtg 60 accggtggtt tgtttgccgg atcaagagctaccaactctt tttccgaagg taactggctt 120 cagcagagcg cagataccaa atactgttcttctagtgtag ccgtagttag gccaccactt 180 caagaactct gtagcaccgc ctacatacctcgctctgcta atcctgttac cagtggctgc 240 tgccagtggc gataagtcgt gtcttacc 26822 259 DNA Artificial Sequence Description of Artificial Sequence DNAencoding origin of replication (ori5′) mutant 5.1 (ori 5.1), a high-copyvariant of ColE1-type plasmid 22 tcttgagatc ctttttttct gcgcgtaatctgctgcttgc aaacaaaaaa accaccgcta 60 ccagcggtgg tttgtttgcc ggatcaagagctttccgaag gtaactggct tcagcagagc 120 gcagatacca aatactgttc ttctagtgtagccgtagtta ggccaccact tcaagaactc 180 tgtagcaccg cctacatacc tcgctctgctaatcctgtta ccagtggctg ctgccagtgg 240 cgataagtcg tgtcttacc 259 23 265 DNAArtificial Sequence Description of Artificial Sequence DNA encodingorigin of replication (ori5′) mutant 5.2 (ori 5.2), a high-copy variantof ColE1-type plasmid 23 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgcaaacaaccac cgctaccagc 60 ggtggtttgt ttgccggatc aagagctacc aactctttttccgaaggtaa ctggcttcag 120 cagagcgcag ataccaaata ctgttcttct agtgtagccgtagttaggcc accacttcaa 180 gaactctgta gcaccgccta catacctcgc tctgctaatcctgttaccag tggctgctgc 240 cagtggcgat aagtcgtgtc ttacc 265 24 263 DNAArtificial Sequence Description of Artificial Sequence DNA encodingorigin of replication (ori5′) mutant 5.3 (ori 5.3), a high-copy variantof ColE1-type plasmid 24 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgcaaacaaaaaa accaccgcgg 60 tggtttgttt gccggatcaa gagctaccaa ctctttttccgaaggtaact ggcttcagca 120 gagcgcagat accaaatact gttcttctag tgtagccgtagttaggccac cacttcaaga 180 actctgtagc accgcctaca tacctcgctc tgctaatcctgttaccagtg gctgctgcca 240 gtggcgataa gtcgtgtctt acc 263 25 112 RNAArtificial Sequence Description of Artificial Sequence RNAII region ofBluescript plasmid 25 gcaaacaaaa aaaccaccgc uaccagcggu gguuuguuugccggaucaag agcuaccaac 60 ucuuuuuccg aagguaacug gcuucagcag agcgcagauaccaaauacug uu 112 26 10 DNA Artificial Sequence Description ofArtificial Sequence first ten nucleotides of SEQ ID NO3 26 ggtttgtttg 1027 11 DNA Artificial Sequence Description of Artificial Sequence 11nucleotide deletion from positions 40-50 of origin mutant 3.4 (ori 3.4)27 caaacaaaaa a 11

What is claimed is:
 1. A ColE1 origin of replication comprising at leastone mutation at one or more nucleotides from position 1 to position 210as determined with reference to SEQ ID NO:1, wherein the mutationincreases plasmid copy number of a plasmid comprising the origin by atleast 2-fold in comparison to a control plasmid comprising the origin ofreplication set forth in SEQ ID NO:1 or confers compatibility with asecond ColE1-type origin.
 2. An origin of replication of claim 1,wherein the origin comprises at least one mutation at one or morenucleotides from position 1 to position 150 as determined with referenceto SEQ ID NO:1.
 3. An origin of replication of claim 1, wherein themutation is a deletion.
 4. An origin of replication of claim 3, whereinthe deletion is 20 or fewer nucleotides in length.
 5. An origin ofreplication of claim 1, wherein the mutation is an insertion.
 6. Anorigin of replication of claim 5, wherein the insertion is 20 or fewernucleotides in length.
 7. An origin of replication of claim 1, whereinthe mutation is a substitution.
 8. An origin of replication of claim 1,wherein the mutation occurs in a region selected from the groupconsisting of positions 1 to 68, positions 40 to 50, positions 57 to 60,positions 25 to 27, positions 59-64, positions 192-194, positions128-134, positions 126-128, positions 61-62, positions 93-103, positions47-51, positions 59-65, and positions 58-63.
 9. The origin ofreplication of claim 1, wherein the origin comprises a sequence as setforth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ IDNO:13.
 10. A circular DNA comprising the origin of replication ofclaim
 1. 11. A plasmid comprising an origin of replication of claim 1,wherein the plasmid copy number is increased at least 2-fold incomparison to a control plasmid comprising the origin of replication setforth in SEQ ID NO:1.
 12. A plasmid comprising the origin of replicationof claim 1, wherein the plasmid is compatible with a second ColE1plasmid.
 13. A cell comprising a circular DNA, wherein the circular DNAmolecule comprises a ColE1-related origin of replication as set forth inclaim
 1. 14. The cell of claim 13, wherein the circular DNA is aplasmid.
 15. The cell of claim 14, wherein the cell comprises a secondplasmid.
 16. The cell of claim 15, wherein the ColE1-related origin ofreplication comprises SEQ ID NO:5 or SEQ ID NO:11, and the secondplasmid comprises a second ColE1-related origin.
 17. A method ofgenerating a plasmid at a high copy number, the method comprising:introducing into a bacterial cell a circular DNA comprising a ColE1-typereplication origin, wherein the replication origin comprises at leastone mutation at one or more nucleotides from position 1 to position 210as determined with reference to SEQ ID NO:1, and culturing the bacterialcell.
 18. The method of claim 17, wherein the origin comprises at leastone mutation at one or more nucleotides form position 1 to position 150as determined with reference to SEQ ID NO:1.
 19. The method of claim 17,wherein the mutation is a deletion.
 20. The method of claim 19, whereinthe deletion is 20 or fewer nucleotides in length.
 21. The method ofclaim 17, wherein the mutation is an insertion.
 22. The method of claim21, wherein the insertion is 20 or fewer nucleotide in length.
 23. Themethod of claim 17, wherein the mutation is a substitution.
 24. Themethod of claim 17, wherein the deletion or insertion occurs within atleast one of the regions selected from the group consisting of positions1 to 68, positions 40 to 50, positions 57 to 60, positions 25 to 27,positions 59-64, positions 192-194, positions 128-134, positions126-128, positions 61-62, positions 93-103, positions 47-51, positions59-65, and positions 58-63.
 25. The method of claim 17, wherein theorigin comprises a sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, or SEQ ID NO:13.
 26. The method of claim 25,wherein the origin comprises SEQ ID NO:5 or SEQ ID NO:11 and the methodfurther comprises introducing into a bacterial cell a circular DNAcomprising a second ColE1-type replication origin.